William Goossens

Cytoplasmic filaments in the crystalline lens of various species: functional correlations.

Abstract The distribution of cytoplasmic filaments in lenses of five species was studied with the electron microscope. Two distinct patterns emerged. One pattern, in which filaments are grouped in characteristic bundles around the nucleus, in processes, and throughout the subcortical cytoplasm of epithelial cells, is typical of spherical, non-accommodating lenses of mice and rats. The second pattern is associated with anteriorly-flattened, accommodating lenses of infant human, squirrel and frog. In these, filaments are scattered in epithelial cells, but are accumulated on either side of the plasma membrane junction between epithelial cells and lens fibers. They are especially dense on the lens fiber side of the junction, and form a lattice associated with the lens fiber plasma membrane. The lattice is less extensive along the sides of lens fibers not in contact with epithelial cells. In spherical lenses the epithelial-fiber lattice is greatly reduced. Filaments in both types of lenses ranged in diameter between 5 and 11 nm. The filaments are thought to be a mixture of thin and intermediate filaments. It is hypothesized that the role of cytoplasmic filaments in lens, depending on the pattern present, is either to structurally support a spherical shape, or to provide a contractile force or elasticity to return the flattened anterior surface to the accommodated state in conjunction with the elasticity of the lens capsule.

IFN-γ regulation of vacuolar pH, cathepsin D processing and autophagy in mammary epithelial cells

In this study we examined the ability of interferon‐γ (IFN‐γ) to regulate mammary epithelial cell growth and gene expression, with particular emphasis on two genes: Maspin (a member of serine protease inhibitor superfamily), and the lysosomal aspartyl endopeptidase cathepsin D (CatD). The protein products of these genes are critically involved in regulation of multitude of biological functions in different stages of mammary tissue development and remodeling. In addition, the expression of Maspin is down‐regulated in primary breast cancer and is lost in metastatic disease, while CatD is excessively produced and aberrantly secreted by breast cancer cells. We report that IFN‐γ receptors are expressed in mammary epithelial cells, and receptor engagement by IFN‐γ transduces the IFN‐γ signal via Stat‐1 resulting in decreased vacuolar pH. This change in vacuolar pH alters CatD protein processing and secretion concurrent with increased Maspin secretion. In addition, IFN‐γ exerts a suppressive effect on cell growth and proliferation, and induces morphological changes in mammary epithelial cells. Our studies also reveal that breast cancer cells, which are devoid of Maspin, are refractory to IFN‐γ with respect to changes in vacuolar pH and CatD. However, Maspin transfection of breast cancer cells partially sensitizes the cells to IFN‐γs effect, thus providing new therapeutic implications. J. Cell. Biochem. 105: 208–218, 2008.

Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies.

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers - OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 - in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3-5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.

Reconstitution of trabecular meshwork GAGs: Influence of hyaluronic acid and chondroitin sulfate on flow rates

Purpose:This study was undertaken to determine whether the concentration of hyaluronic acid (HA) and of chondroitin sulfate (CS) occurring in the normal and the primary open-angle glaucoma (POAG) trabecular meshwork (TM) influences flow rates in vitro as a function of pressure. Methods:We tested 100, 500, and 4000 kDa molecular weight HA, CS, reconstituted normal and POAG TM HA-CS and juxtacanalicular connective tissue (JCT) HA-CS in a micro test chamber to determine initial and steady-state flow rates. The resistance and permeability (Ko) were calculated; Linear Newtonian mechanics were used to determine the possible contributions of the hydrophobic interactions of HA. Results:Initial flow rates increased in the pressure range of 5 to 20 mm Hg for the three HA preparations and the flow rates declined in the pressure range of 20 to 40 mm Hg. Flow rates of reconstituted normal TM and JCT were optimum at 10 mm Hg and then declined with increasing pressure. Flow rates of reconstituted POAG TM and JCT were optimum only at 5 mm Hg and then declined. The steady-state rate of POAG JCT HA-CS at 10 mm Hg was slow: the transition time (ie, the time required to start an increase in flow rate) was 29 hours and the lag time (ie, the time required to obtain steady-state flow rate) was 17 hours. The maximum flow rate in POAG JCT HA-CS decreased by 37.2% from the normal JCT HA-CS. The calculated resistance of reconstituted POAG JCT HA-CS was approximately 18% of the total resistance of the human JCT compared with 10% in the normal JCT. Conclusions:Hyaluronic acid and CS contribute to flow resistance and influence flow rate in vitro. The influence of HA is particularly sensitive to an increase in the pressure gradient, which may be caused by unfolding of the hydrophobic interactions of HA polymers that further entangles the HA polymer. The POAG JCT HA-CS concentrations represent a significant factor in outflow resistance in POAG, particularly at higher pressures.

Pathological changes in exposed neural tissue of fetal delayed splotch (Spd) mice

If meningomyelocele is indeed a progressive intrauterine process, then early delivery or possibly intrauterine repair of meningomyelocele becomes an issue. Utilizing the delayed splotch (Spd) mouse, a genetically transmitted neural tube defect model, we looked for evidence of abnormalities of neural tissue exposed to amniotic fluid. Affected embryonic and fetal mice were examined with the light microscope, and also with the transmission and scanning electron microscope. Neuronal development and programmed cell death paralleled normal fetal development. No evidence of inflammation on or within the exposed neural tissue was observed. Because the vascular supply to the alar and basilar plate are different, vascular development was also examined and no difference could be found. In conclusion, we found no evidence of deterioration of the exposed neural tube during the gestational period of a mouse, which suggests that exposure of unneurulated spinal cord to amniotic fluid is not a risk factor to the fetus with a neural tube defect.

Cleavage of Histone 3 by Cathepsin D in the Involuting Mammary Gland

The post-lactational regression of mammary gland is a complex multi-step process designed to conserve the biological function of the gland for next pregnancy. This developmental stage is a biological intrigue with great relevance to breast cancer research, and thus has been the subject of intensive scrutiny. Multipronged studies (microarray, proteomics profiling, animal knock-out models) have provided a repertoire of genes critical to involution. However, the caveat of these approaches remains in their failure to reveal post-translational modification(s), an emerging and critical aspect of gene regulation in developmental processes and mammary gland remodeling. The massive surge in the lysosomal enzymes concurrent with the onset of involution has been known for decades, and considered essential for “clearance” purposes. However, functional significance of these enzymes in diverse biological processes distinct from their proteolytic activity is just emerging. Studies from our laboratory had indicated specific post-translational modifications of the aspartyl endopeptidase Cathepsin D (CatD) at distinct stages mammary gland development. This study addresses the biological significance of these modifications in the involution process, and reveals that post-translational modifications drive CatD into the nucleus to cleave Histone 3. The cleavage of Histone 3 has been associated with cellular differentiation and could be critical instigator of involution process. From functional perspective, deregulated expression and increased secretion of CatD are associated with aggressive and metastatic phenotype of breast cancer. Thus unraveling CatD’s physiological functions in mammary gland development will bridge the present gap in understanding its pro-tumorigenic/metastatic functions, and assist in the generation of tailored therapeutic approaches.

The presence of transcription factors in fetal bovine sera.

SummaryThree sources of fetal bovine serum (FBS) were fractionated by ammonium sulfate precipitation and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Immobilon-P membranes, immunoblotted with a panel of transcription factor antibodies, and detected by enhanced chemiluminescence. Nine transcription factors were detected—ATF-2, SRE-ZBP, GATA-2, TFIID, Ets-1/Ets-2, E2F-1, Oct-2, p53, and AP-2; four transcription factors were not detected—Myo D, CREB, Sp2, and Wilms’ tumor. The results indicated the presence of varying amounts of several transcription factors in three commercial sources and may represent heretofore unrecognized factors influencing cell culture.

THE PRESENCE OF TRANSCRIPTION FACTORS IN CHICKEN ALBUMIN, YOLK AND BLASTODERM

SummaryEmbryonic development is determined by preset intrinsic programs and extrinsic signals. To explore the possibility that transcription factors are present at the onset of development, preparations of yolk, albumin, and blastoderm from unfertilized and fertilized white Leghorn chicken eggs were screened by a panel of 16 transcription factor antibodies with Western blot techniques. Yolk was positive for 13 transcription factors, whereas blastoderm was positive for 10, and albumin was positive for 5. In yolk, several transcription factors, GATA-2, E2F-1, MyoD, and TFIID, were developmentally regulated. These results indicate that intracellular yolk and extracellular albumin contain transcription factors which presumably influence early chick embryonic development from prefertilization to the late blastoderm stage. Thus, the utility of preset maternal transcription factors within yolk and albumin complement maternally derived mRNA to determine the early development of the zygote.

Ultrastructural alterations in the aqueous outflow pathway of adult buphthalmic rabbits

The aqueous outflow pathway of adult rabbit eyes with congenital glaucoma (buphthalmos) was examined by light microscopy and by scanning and transmission electron microscopy. The morphology of the buphthalmic rabbit aqueous outflow pathway was markedly abnormal when examined at 6 months, 1 yr, and 2 yr displaying apparent loss and/or compression of the iris pillars, dilation of the intertrabecular spaces, loss of endothelial cell-to-cell association and disorganization of trabecular lamellae, and posterior displacement of the aqueous plexus. In addition, the trabecular meshwork lamellae were observed only adjacent to the sclera and the inner portion of the trabecular meshwork was limited to swirls of collagen with scattered cells. These morphological findings suggest that the disease process in the rabbit principally involves an alteration in the differentiation and maintenance of the structural integrity of the trabecular meshwork. The loss of structural support of the buphthalmic trabecular meshwork may be a factor in the wide variation in intraocular pressure and may allow for compression of the trabecular meshwork against the aqueous plexus.

Ultrastructural Studies of Primary Congenital Glaucoma in Rabbits

BACKGROUND The cause of congenital glaucoma is unknown. METHODS To determine whether the site of impaired aqueous outflow is the entrance to the trabecular meshwork (TM), within the TM, the aqueous drainage plexus, or a combination thereof, the process of TM development was examined by scanning and transmission electron microscopy on postnatal day 3 and weeks 1, 2, 3, 4, and 6 in New Zealand rabbits homozygous for the buphthalmic (bu/bu) gene compared with age-matched controls. RESULTS Openings to the entrance of the TM in congenital glaucoma were observed, and there was no evidence of an endothelial membrane occluding aqueous flow to the TM. The morphology of the congenital glaucoma TM was abnormal in all bu/bu rabbits by 2 weeks and was characterized by a smaller entrance to the TM at the iris base, smaller intertrabecular openings within and between the trabecular lamellae, and at 6 weeks, iris pillars with extensive lateral extensions in the angle recess. Most intertrabecular spaces were open, however, the inner intertrabecular spaces adjacent to the aqueous plexus were compressed. CONCLUSION These results suggest the development of congenital glaucoma, which involves a mutation in an autosomal recessive gene and leads to loss of function of a gene(s) required for the differentiation of the TM.